Involvement of XZFP36L1,an RNA-binding protein,in Xenopus neural development

Xenopus ZFP36L1(zinc finger protein 36,C3H type-like 1)belongs to the ZFP36 family of RNA-binding proteins,which contains two characteristic tandem CCCH-type zinc-finger domains.The ZFP36 proteins can bind AU-rich elements in 3'untranslated regions of target mRNAs and promote their turnover.However,the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely unknown.The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain,forebrain-midbrain boundary,and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development.Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation,leading to severe neural tube defects.The function of XZP36L1 requires both its zinc finger and C terminal domains,which also affect its subcellular localization.These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

Involvement of XZFP36L1, an RNA-binding protein, in Xenopus neural development ＇

Xenopus ZFP36L1 （zinc finger protein 36, C3H type-like 1） belongs to the ZFP36 family of RNA-binding proteins, which contains two characteristic tandem CCCH-type zinc-finger domains. The ZFP36 proteins can bind AU-rich elements in 3＇ untranslated regions of target mRNAs and promote their turnover. However, the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely unknown. The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain, forebrain-midbrain boundary, and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development. Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation, leading to severe neural tube defects. The function of XZP36L1 requires both its zinc finger and C terminal domains, which also affect its subcellular localization. These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

Foreword

The State Key Laboratory of Genetic Resources and Evolution,located at the Kunming Institute of Zoology,Chinese Academy of Sciences(CAS),is a leading research center in genetic resource conservation and evolutionary biology.In this special issue of Zoological Research on“Animal Genetic diversity,development and evolution”,we have compiled 12 review and research articles from the Key Lab as well as 4 related articles from external authors,covering,among other topics,genetic biodiversity,molecular phylogeny,evolution of gene families,and the use of mitochondrial DNA in the study of adaptive evolution and tumor evolution.

Single-cell RNA Sequencing Reveals Thoracolumbar Vertebra Heterogeneity and Rib-genesis in Pigs

Development of thoracolumbar vertebra(TLV)and rib primordium(RP)is a common evolutionary feature across vertebrates,although whole-organism analysis of the expression dynamics of TLV-and RP-related genes has been lacking.Here,we investigated the single-cell transcriptome landscape of thoracic vertebra(TV),lumbar vertebra(LV),and RP cells from a pig embryo at 27 days post-fertilization(dpf)and identified six cell types with distinct gene expression signatures.In-depth dissection of the gene expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and RP development.Further analysis of cell type-specific and strand-specific expression uncovered the extremely high level of HOXA103′-UTR sequence specific to osteoblasts of LV cells,which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition.Thus,this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell type-specific resolution,which serves as a comprehensive view on the transcriptional profile of animal embryo development.

ZC4H2 stabilizes RNF220 to pattern ventral spinal cord through modulating Shh/Gli signaling

ZC4H2 encodes a C4H2 type zinc-finger nuclear factor,the mutation of which has been associated with disorders with various clinical phenotypes in human,including developmental delay,intellectual disability and dystonia.ZC4H2 has been suggested to regulate spinal cord patterning in zebrafish as a co-factor for RNF220,an ubiquitin E3 ligase involved in Gli signaling.Here we showed that ZC4H2 and RNF220 knockout animals phenocopy each other in spinal patterning in both mouse and zebrafish,with mispatterned progenitor and neuronal domains in the ventral spinal cord.We showed evidence that ZC4H2 is required for the stability of RNF220 and also proper Gli ubiquitination and signaling in vivo.Our data provides new insights into the possible etiology of the neurodevelopmental impairments observed in ZC4H2-associated syndromes.

Sequential stabilization of RNF220 by RLIM and ZC4H2 during cerebellum development and Shh-group medulloblastoma progression

Sonic hedgehog (Shh) signaling is essential for the proliferation of cerebellar granule neuron progenitors (CGNPs), and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma (MB). During vertebrate neural development, RNF220, a ubiquitin E3 ligase, is involved in spinal cord patterning by modulating the subcellular location of glioma-associated oncogene homologs (Glis) through ubiquitination. RNF220 is also required for full activation of Shh signaling during cerebellum development in an epigenetic manner through targeting embryonic ectoderm development. ZC4H2 was reported to be involved in spinal cord patterning by acting as an RNF220 stabilizer. Here, we provided evidence to show that ZC4H2 is also required for full activation of Shh signaling in CGNP and MB progression by stabilizing RNF220. In addition, we found that the ubiquitin E3 ligase RING finger LIM domain-binding protein (RLIM) is responsible for ZC4H2 stabilization via direct ubiquitination, through which RNF220 is also thus stabilized. RLIM is a direct target of Shh signaling and is also required for full activation of Shh signaling in CGNP and MB cell proliferation. We further provided clinical evidence to show that the RLIM‒ZC4H2‒RNF220 cascade is involved in Shh-group MB progression. Disease-causative human RLIM and ZC4H2 mutations affect their interaction and regulation. Therefore, our study sheds light on the regulation of Shh signaling during cerebellar development and MB progression and provides insights into neural disorders caused by RLIM or ZC4H2 mutations.

Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes

The secreted Wnt signaling inhibitor Dickkopfl （Dkkl） plays key role in vertebrate head induction. Its receptor Kremen synergizes with Dkkl in Wnt inhibition. Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus （Branchiostoma belcheri）. During embryonic and larval development, BbDkkl/2/4 is expressed in the posterior mesoendoderm, anterior somatic mesoderm and the pharyngeal regions. Its expression becomes restricted to the pharyngeal region on the left side at larval stages. In 45 h larvae, BbDkkl/2/4 is expressed specifically in the cerebral vesicle. BbDkk3 was only detected at larval stages in the mid-intestine region. Seven Kremen related genes were identified in the genome of the Florida amphioxus （Branchiostoma floridae）, clustered in 4 scaffolds, and are designated Kremenl-4 and Kremen-like 1-3, respectively. In B. belcheri, Kremenl is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region. In luciferase reporter assays, BbDkkl/2/4 can strongly inhibit Wnt signaling, while BbDkk3, BbKremenl and BbKremen3 can not. No co-operative effect was observed between amphioxus Dkkl/2/4 and Kremens, suggesting that the interaction between Dkk and Kremen likely originated later during evolution.

Origin of new genes after zygotic genome activation in vertebrate

New genes are drivers of evolutionary innovation and phenotypic evolution. Expression of new genes in early development raises the possibility that new genes could originate and be recruited for functions in embryonic development, but this remains undocu- mented. Here, based on temporal gene expression at different developmental stages in Xenopus tropicolis, we found that young protein-coding genes were significantly enriched for expression in developmental stages occurring after the midblastula trans- ition （MBT）, and displayed a decreasing trend in abundance in the subsequent stages after MBT. To complement the finding, we demonstrate essential functional attributes of a young orphan gene, named as Fog2, in morphological development. Our data indicate that new genes could originate after MBT and be recruited for functions in embryonic development, and thus provide insights for better understanding of the origin, evolution, and function of new genes.

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes

The evolution of the central nervous system（CNS） is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties,gene duplication might play an important role in the functional innovation of vertebrate CNS.In this study,we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution.We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri.Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos,and more than 50%and 30%duplicate genes are expressed in the telencephalon and mid-hindbrain boundary,respectively,which are mostly considered as two innovations in the vertebrate CNS.Interestingly,more than 50%of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization,indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates.Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes.

Haploinsufficiency oftheTDP43 ubiquitin E3 ligase RNF220 leads to ALS-like motor neuron defects in the mouse

TDP43 pathology is seen in a large majority of amyotrophic lateral sclerosis(ALS)cases,suggesting a central pathogenic role of this regulatory protein.Clarifying the molecular mechanism controlling TDP43 stability and subcellular location might provide important insights into ALS therapy.The ubiquitin E3 ligase RNF220 is involved in different neural developmental processes through various molecular targets in the mouse.Here,we report that the RNF2207 mice showed progressively decreasing mobility to different extents,some of which developed typical ALS pathological characteristics in spinal motor neurons,including TDP43 cytoplasmic accumulation,atrocytosis,muscle denervation,and atrophy.Mechanistically,RNF220 interacts with TDP43 in vitro and in vivo and promotes its polyubiquitination and proteasomal degradation.In conclusion,we propose that RNF220 might be a modifier of TDP43 function in vivo and contribute to TDP43 pathology in neurodegenerative disease like ALS.